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Diabetic encephalopathy (DE) is an inflammation-associated diabetes mellitus (DM) complication. Inflammation and coagulation are linked and are both potentially modulated by inhibiting the thrombin cellular protease-activated receptor 1 (PAR1). Our aim was to study whether coagulation pathway modulation affects DE. Diabetic C57BL/6 mice were treated with PARIN5, a novel PAR1 modulator. Behavioral changes in the open field and novel object recognition tests, serum neurofilament (NfL) levels and thrombin activity in central and peripheral nervous system tissue (CNS and PNS, respectively), brain mRNA expression of tumor necrosis factor α (TNF-α), Factor X (FX), prothrombin, and PAR1 were assessed. Subtle behavioral changes were detected in diabetic mice. These were accompanied by an increase in serum NfL, an increase in central and peripheral neural tissue thrombin activity, and TNF-α, FX, and prothrombin brain intrinsic mRNA expression. Systemic treatment with PARIN5 prevented the appearance of behavioral changes, normalized serum NfL and prevented the increase in peripheral but not central thrombin activity. PARIN5 treatment prevented the elevation of both TNF-α and FX but significantly elevated prothrombin expression. PARIN5 treatment prevents behavioral and neural damage in the DE model, suggesting it for future clinical research. [ABSTRACT FROM AUTHOR]

Background. Due to the interactions between neuroinflammation and coagulation, the neural effects of lipopolysaccharide (LPS)-induced inflammation (1 mg/kg, intraperitoneal (IP), n = 20) and treatment with the anti-thrombotic enoxaparin (1 mg/kg, IP, 15 min, and 12 h following LPS, n = 20) were studied in C57BL/6J mice. Methods. One week after LPS injection, sensory, motor, and cognitive functions were assessed by a hot plate, rotarod, open field test (OFT), and Y-maze. Thrombin activity was measured with a fluorometric assay; hippocampal mRNA expression of coagulation and inflammation factors were measured by real-time-PCR; and serum neurofilament-light-chain (NfL), and tumor necrosis factor-α (TNF-α) were measured by a single-molecule array (Simoa) assay. Results. Reduced crossing center frequency was observed in both LPS groups in the OFT (p = 0.02), along with a minor motor deficit between controls and LPS indicated by the rotarod (p = 0.057). Increased hippocampal thrombin activity (p = 0.038) and protease-activated receptor 1 (PAR1) mRNA (p = 0.01) were measured in LPS compared to controls, but not in enoxaparin LPS-treated mice (p = 0.4, p = 0.9, respectively). Serum NfL and TNF-α levels were elevated in LPS mice (p < 0.05) and normalized by enoxaparin treatment. Conclusions. These results indicate that inflammation, coagulation, neuronal damage, and behavior are linked and may regulate each other, suggesting another pharmacological mechanism for intervention in neuroinflammation. [ABSTRACT FROM AUTHOR]

Thrombin is present in peripheral nerves and is involved in the pathogenesis of neuropathy. We evaluated thrombin activity in skin punch biopsies taken from the paws of male mice and rats and from the legs of patients with suspected small-fiber neuropathy (SFN). In mice, inflammation was induced focally by subcutaneous adjuvant injection to one paw and systemically by intraperitoneal lipopolysaccharides (LPS) administration. One day following injection, thrombin activity increased in the skin of the injected compared with the contralateral and non-injected control paws (p = 0.0009). One week following injection, thrombin increased in both injected and contralateral paws compared with the controls (p = 0.026), coupled with increased heat-sensitivity (p = 0.009). Thrombin activity in the footpad skin was significantly increased one week after systemic administration of LPS compared with the controls (p = 0.023). This was not accompanied by increased heat sensitivity. In human skin, a correlation was found between nerve fiber density and thrombin activity. In addition, a lower thrombin activity was measured in patients with evidence of systemic inflammation compared with the controls (p = 0.0035). These results support the modification of skin thrombin activity by regional and systemic inflammation as well as a correlation with nerve fiber density. Skin thrombin activity measurments may aid in the diagnosis and treatment of SFN. [ABSTRACT FROM AUTHOR]

The aim of this study was to characterize the distribution of the thrombin receptor, protease activated receptor 1 (PAR1), in the neuroretina. Neuroretina samples of wild-type C57BL/6J and PAR1−/− mice were processed for indirect immunofluorescence and Western blot analysis. Reverse transcription quantitative real-time PCR (RT-qPCR) was used to determine mRNA expression of coagulation Factor X (FX), prothrombin (PT), and PAR1 in the isolated neuroretina. Thrombin activity following KCl depolarization was assessed in mouse neuroretinas ex vivo. PAR1 staining was observed in the retinal ganglion cells, inner nuclear layer cells, and photoreceptors in mouse retinal cross sections by indirect immunofluorescence. PAR1 co-localized with rhodopsin in rod outer segments but was not expressed in cone outer segments. Western blot analysis confirmed PAR1 expression in the neuroretina. Factor X, prothrombin, and PAR1 mRNA expression was detected in isolated neuroretinas. Thrombin activity was elevated by nearly four-fold in mouse neuroretinas following KCl depolarization (0.012 vs. 0.044 mu/mL, p = 0.0497). The intrinsic expression of coagulation factors in the isolated neuroretina together with a functional increase in thrombin activity following KCl depolarization may suggest a role for the PAR1/thrombin pathway in retinal function. [ABSTRACT FROM AUTHOR]

Axonal and neuronal pathologies are a central constituent of multiple sclerosis (MS) and its animal model, experimental autoimmune encephalomyelitis (EAE), induced by the myelin oligodendrocyte glycoprotein (MOG) 35–55 peptide. In this study, we investigated neurodegenerative manifestations in chronic MOG 35–55 induced EAE and the effect of glatiramer acetate (GA) treatment on these manifestations. We report that the neuronal loss seen in this model is not attributed to apoptotic neuronal cell death. In EAE-affected mice, axonal damage prevails from the early disease phase, as revealed by analysis of neurofilament light (NFL) leakage into the sera along the disease duration, as well as by immunohistological examination. Elevation of interstitial glutamate concentrations measured in the cerebrospinal fluid (CSF) implies that glutamate excess plays a role in the damage processes inflicted by this disease. GA applied as a therapeutic regimen to mice with apparent clinical symptoms significantly reduces the pathological manifestations, namely apoptotic cell death, NFL leakage, histological tissue damage, and glutamate excess, thus corroborating the neuroprotective consequences of this treatment. [ABSTRACT FROM AUTHOR]

Complement and coagulation are both key systems that defend the body from harm. They share multiple features and are similarly activated. They each play individual roles in the systemic circulation in physiology and pathophysiology, with significant crosstalk between them. Components from both systems are mapped to important structures in the central nervous system (CNS) and peripheral nervous system (PNS). Complement and coagulation participate in critical functions in neuronal development and synaptic plasticity. During pathophysiological states, complement and coagulation factors are upregulated and can modulate synaptic transmission and neuronal conduction. This review summarizes the current evidence regarding the roles of the complement system and the coagulation cascade in the CNS and PNS. Possible crosstalk between the two systems regarding neuroinflammatory-related effects on synaptic transmission and neuronal conduction is explored. Novel treatment based on the modulation of crosstalk between complement and coagulation may perhaps help to alleviate neuroinflammatory effects in diseased states of the CNS and PNS. [ABSTRACT FROM AUTHOR]

Diabetic peripheral neuropathy (DPN) is a disabling common complication of diabetes mellitus (DM). Thrombin, a coagulation factor, is increased in DM and affects nerve function via its G-protein coupled protease activated receptor 1 (PAR1). Methods: A novel PAR1 modulator (PARIN5) was designed based on the thrombin PAR1 recognition site. Coagulation, motor and sensory function and small fiber loss were evaluated by employing the murine streptozotocin diabetes model. Results: PARIN5 showed a safe coagulation profile and showed no significant effect on weight or glucose levels. Diabetic mice spent shorter time on the rotarod (p < 0.001), and had hypoalgesia (p < 0.05), slow conduction velocity (p < 0.0001) and reduced skin innervation (p < 0.0001). Treatment with PARIN5 significantly improved rotarod performance (p < 0.05), normalized hypoalgesia (p < 0.05), attenuated slowing of nerve conduction velocity (p < 0.05) and improved skin innervation (p <0.0001). Conclusion: PARIN5 is a novel pharmacological approach for prevention of DPN development, via PAR1 pathway modulation. [ABSTRACT FROM AUTHOR]

Glia cells are involved in upper motor neuron degeneration in amyotrophic lateral sclerosis (ALS). Protease activated receptor 1 (PAR1) pathway is related to brain pathologies. Brain PAR1 is located on peri-synaptic astrocytes, adjacent to pyramidal motor neurons, suggesting possible involvement in ALS. Brain thrombin activity in superoxide dismutase 1 (SOD1) mice was measured using a fluorometric assay, and PAR1 levels by western blot. PAR1 was localized using immunohistochemistry staining. Treatment targeted PAR1 pathway on three levels; thrombin inhibitor TLCK (N-Tosyl-Lys-chloromethylketone), PAR1 antagonist SCH-79797 and the Ras intracellular inhibitor FTS (S-trans-trans-farnesylthiosalicylic acid). Mice were weighed and assessed for motor function and survival. SOD1 brain thrombin activity was increased (p < 0.001) particularly in the posterior frontal lobe (p = 0.027) and hindbrain (p < 0.01). PAR1 levels were decreased (p < 0.001, brain, spinal cord, p < 0.05). PAR1 and glial fibrillary acidic protein (GFAP) staining decreased in the cerebellum and cortex. SOD1 mice lost weight (≥17 weeks, p = 0.047), and showed shorter rotarod time (≥14 weeks, p < 0.01). FTS 40mg/kg significantly improved rotarod scores (p < 0.001). Survival improved with all treatments (p < 0.01 for all treatments). PAR1 antagonism was the most efficient, with a median survival improvement of 10 days (p < 0.0001). Our results support PAR1 pathway involvement in ALS. [ABSTRACT FROM AUTHOR]

Background: Neural inflammation is linked to coagulation. Low levels of thrombin have a neuroprotective effect, mediated by activated protein C (APC). We describe a sensitive novel method for the measurement of APC activity at the low concentrations found in neural tissue. Methods: APC activity was measured using a fluorogenic substrate, Pyr-Pro-Arg-AMC, cleaved preferentially by APC. Selectivity was assessed using specific inhibitors and activators. APC levels were measured in human plasma, in glia cell lines, in mice brain slices following mild traumatic brain injury (mTBI) and systemic lipopolysaccharide (LPS) injection, and in cerebrospinal fluid (CSF) taken from viral meningoencephalitis patients and controls. Results: Selectivity required apixaban and alpha-naphthylsulphonylglycyl-4-amidinophenylalanine piperidine (NAPAP). APC levels were easily measurable in plasma and were significantly increased by Protac and CaCl2. APC activity was significantly higher in the microglial compared to astrocytic cell line and specifically lowered by LPS. Brain APC levels were higher in posterior regions and increased by mTBI and LPS. Highly elevated APC activity was measured in viral meningoencephalitis patients CSF. Conclusions: This method is selective and sensitive for the measurement of APC activity that significantly changes during inflammation in cell lines, animal models and human CSF. [ABSTRACT FROM AUTHOR]

Apolipoprotein E (APOE) ε4 gene allele and type 2 diabetes mellitus (T2DM) are prime risk factors for Alzheimer's disease (AD). Despite evidence linking T2DM and apoE4, the mechanism underlying their interaction is yet to be determined. In the present study, we employed a model of APOE-targeted replacement mice and high-fat diet (HFD)-induced insulin resistance to investigate diabetic mechanisms associated with apoE4 pathology and the extent to which they are driven by peripheral and central processes. Results obtained revealed an intriguing pattern, in which under basal conditions, apoE4 mice display impaired glucose and insulin tolerance and decreased insulin secretion, as well as cognitive and sensorimotor characteristics relative to apoE3 mice, while the HFD impairs apoE3 mice without significantly affecting apoE4 mice. Measurements of weight and fasting blood glucose levels increased in a time-dependent manner following the HFD, though no effect of genotype was observed. Interestingly, sciatic electrophysiological and skin intra-epidermal nerve fiber density (IENFD) peripheral measurements were not affected by the APOE genotype or HFD, suggesting that the observed sensorimotor and cognitive phenotypes are related to central nervous system processes. Indeed, measurements of hippocampal insulin receptor and glycogen synthase kinase-3β (GSK-3β) activation revealed a pattern similar to that obtained in the behavioral measurements while Akt activation presented a dominant effect of diet. HFD manipulation induced genotype-independent hyperlipidation of apoE, and reduced levels of brain apoE in apoE3 mice, rendering them similar to apoE4 mice, whose brain apoE levels were not affected by the diet. No such effect was observed in the peripheral plasma levels of apoE, suggesting that the pathological effects of apoE4 under the control diet and apoE3 under HFD conditions are related to the decreased levels of brain apoE. Taken together, our data suggests that diabetic mechanisms play an important role in mediating the pathological effects of apoE4 and that consequently, diabetic-related therapy may be useful in treating apoE4 pathology in AD. [ABSTRACT FROM AUTHOR]